An In Vitro System to Measure the Positioning, Stiffness, and Rupture of the Nucleus in Skeletal Muscle

The murine alpha B-crystallin gene was cloned and its expression was examined. In the mouse, significant levels of alpha B-crystallin RNA were detected not only in lens but also in heart, skeletal muscle, kidney, and lung; low and trace levels were detected in brain and spleen, respectively. The RNA species in lung, brain, and spleen was 400 to 500 bases larger than that in the other tissues. Transcription in lens, heart, skeletal muscle, kidney, and brain initiated at the same position. A mouse alpha B-crystallin mini-gene was constructed and was introduced into the germ line of mice, and its expression was demonstrated to parallel that of the endogenous gene. Transgene RNA was always detected in lens, heart, and skeletal muscle, while expression in kidney and lung was variable; it remains uncertain whether there is transgene expression in brain and spleen. These results demonstrate that regulatory sequences controlling expression of the alpha B-crystallin gene lie between sequences 666 base pairs upstream of the transcription initiation site and 2.4 kilobase pairs downstream of the poly(A) addition site and are not located within the introns. Transfection studies with a series of alpha B-crystallin mini-gene deletion mutants revealed that sequences between positions -222 and -167 were required for efficient expression in primary embryonic chick lens cells; sequences downstream of the poly(A) addition signal were dispensable for expression in this in vitro system.

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The in vitro effect of corticosterone on insulin binding and glucose metabolism in mouse skeletal muscles

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y85-185 ◽

1985 ◽

Vol 63 (9) ◽

pp. 1133-1138 ◽

Cited By ~ 10

Author(s):

M. H. Tan ◽

A. Bonen

Keyword(s):

Skeletal Muscle ◽

Skeletal Muscles ◽

Extensor Digitorum Longus ◽

Insulin Binding ◽

In Vivo Model ◽

In Vitro System ◽

Vitro Effect ◽

Contralateral Muscle

We studied the in vitro effect of corticosterone on insulin binding, uptake of 2-deoxy-D-glucose, glycolysis, and glycogenesis in the soleus and extensor digitorum longus (EDL) of Swiss–Webster mice. In each experiment, one muscle (soleus/EDL) was incubated with corticosterone (0.1, 1, 50, and 100 μg/mL) and the respective contralateral muscle was incubated without corticosterone, but at the same insulin and pH levels. Corticosterone did not affect insulin binding in both muscles. However, corticosterone decreased the uptake of 2-deoxy-D-glucose and the rate of glycolysis and glycogenesis in both muscles when the dose was pharmacologic (50 and 100 μg/mL), but not when it was physiologic (0.1 and 1 μg/mL). For glycolysis and glycogenesis, the suppression was greater in the EDL when compared with the soleus. This suppression was seen in both basal and insulin-stimulated conditions. In this in vitro system, where the experimental muscle is not exposed to prior hyperinsulinemia as in the in vivo model, corticosterone, at pharmacologic doses, affects postreceptor events without altering the insulin binding in the skeletal muscle.

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Expression of the murine alpha B-crystallin gene is not restricted to the lens

Molecular and Cellular Biology ◽

10.1128/mcb.9.3.1083-1091.1989 ◽

1989 ◽

Vol 9 (3) ◽

pp. 1083-1091

Author(s):

R A Dubin ◽

E F Wawrousek ◽

J Piatigorsky

Keyword(s):

Skeletal Muscle ◽

Transcription Initiation ◽

Germ Line ◽

Initiation Site ◽

Regulatory Sequences ◽

Base Pairs ◽

In Vitro System ◽

Endogenous Gene ◽

Efficient Expression

The murine alpha B-crystallin gene was cloned and its expression was examined. In the mouse, significant levels of alpha B-crystallin RNA were detected not only in lens but also in heart, skeletal muscle, kidney, and lung; low and trace levels were detected in brain and spleen, respectively. The RNA species in lung, brain, and spleen was 400 to 500 bases larger than that in the other tissues. Transcription in lens, heart, skeletal muscle, kidney, and brain initiated at the same position. A mouse alpha B-crystallin mini-gene was constructed and was introduced into the germ line of mice, and its expression was demonstrated to parallel that of the endogenous gene. Transgene RNA was always detected in lens, heart, and skeletal muscle, while expression in kidney and lung was variable; it remains uncertain whether there is transgene expression in brain and spleen. These results demonstrate that regulatory sequences controlling expression of the alpha B-crystallin gene lie between sequences 666 base pairs upstream of the transcription initiation site and 2.4 kilobase pairs downstream of the poly(A) addition site and are not located within the introns. Transfection studies with a series of alpha B-crystallin mini-gene deletion mutants revealed that sequences between positions -222 and -167 were required for efficient expression in primary embryonic chick lens cells; sequences downstream of the poly(A) addition signal were dispensable for expression in this in vitro system.

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Ultrastructural autoradiography of L6 skeletal-muscle cells exposed to a tritiated macrolide antibiotic (LY237216) in vitro

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100123465 ◽

1992 ◽

Vol 50 (1) ◽

pp. 612-613

Author(s):

S.L. White ◽

C.B. Jensen ◽

D.D. Giera ◽

D.A. Laska ◽

M.N. Novilla ◽

...

Keyword(s):

Skeletal Muscle ◽

Quantitative Analysis ◽

Macrolide Antibiotic ◽

Circle Method ◽

Apical Surface ◽

Polycarbonate Membrane ◽

L6 Cells ◽

Low Viscosity ◽

Erythromycin A

In vitro exposure to LY237216 (9-Deoxo-11-deoxy-9,11-{imino[2-(2-methoxyethoxy)ethylidene]-oxy}-(9S)-erythromycin), a macrolide antibiotic, was found to induce cytoplasmic vacuolation in L6 skeletal muscle myoblast cultures (White, S.L., unpubl). The present study was done to determine, by autoradiographic quantitative analysis, the subcellular distribution of 3H-LY237216 in L6 cells.L6 cells (ATCC, CRL 1458) were cultured to confluency on polycarbonate membrane filters (Millipore Corp., Bedford, MA) in M-199 medium (GIBCO® Labs) with 10% fetal bovine serum. The cells were exposed from the apical surface for 1-hour to unlabelled-compound (0 μCi/ml) or 50 (μCi/ml of 3H-LY237216 at a compound concentration of 0.25 mg/ml. Following a rapid rinse in compound-free growth medium, the cells were slam-frozen against a liquid nitrogen cooled, polished copper block in a CF-100 cryofixation unit (LifeCell Corp., The Woodlands, TX). Specimens were dried in the MDD-C Molecular Distillation Drier (LifeCell Corp.), vapor osmicated and embedded in Spurrs low viscosity resin. Ultrathin sections collected on formvar coated stainless steel grids were counter-stained, then individually mounted on corks. A monolayer of Ilford L4 nuclear emulsion (Polysciences, Inc., Warrington, PA) was placed on the sections, utilizing a modified “loop method”. The emulsions were exposed for 7-weeks in a light-tight box at 4°C. Autoradiographs were developed in Microdol-X developer and examined on a Philips EM410LS transmission electron microscope. Quantitative analysis of compound localization employed the point and circle approach of Williams; incorporating the probability circle method of Salpeter and McHenry.

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In Vitro Assay of Platelet Adhesiveness with Washed and Tanned Human Red Cells

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651281 ◽

1968 ◽

Vol 20 (03/04) ◽

pp. 384-396 ◽

Cited By ~ 1

Author(s):

G Zbinden ◽

S Tomlin

Keyword(s):

Platelet Adhesion ◽

Sodium Citrate ◽

Blood Platelets ◽

In Vitro Assay ◽

Red Cells ◽

Platelet Adhesiveness ◽

Vitro Assay ◽

In Vitro System ◽

Human Red Cells

SummaryAn in vitro system is described in which adhesion of blood platelets to washed and tannic acid-treated red cells was assayed quantitatively by microscopic observation. ADP, epinephrine and TAME produced a reversible increase in platelet adhesiveness which was antagonized by AMP. With Evans blue, polyanetholsulfonate, phthalanilide NSC 38280, thrombin and heparin at concentrations above 1-4 u/ml the increase was irreversible. The ADP-induced increase in adhesiveness was inhibited by sodium citrate, EDTA, AMP, ATP and N-ethylmaleimide. EDTA, AMP and the SH-blocker N-ethylmaleimide also reduced spontaneous platelet adhesion to red cells. No significant effects were observed with adenosine, phenprocoumon, 5-HT, phthalanilide NSC 57155, various estrogens, progestogens and fatty acids, acetylsalicylic acid and similarly acting agents, hydroxylamine, glucose and KCN. The method may be useful for the screening of thrombogenic and antithrombotic properties of drugs.

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The Mesothelial Cell as a Non-Thrombogenic Surface

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661149 ◽

1984 ◽

Vol 52 (02) ◽

pp. 102-104 ◽

Cited By ~ 25

Author(s):

L J Nicholson ◽

J M F Clarke ◽

R M Pittilo ◽

S J Machin ◽

N Woolf

Keyword(s):

Electron Microscopy ◽

Scanning Electron Microscopy ◽

Blood Flow ◽

Cell Surface ◽

Mesothelial Cell ◽

Mesothelial Cells ◽

Plastic Film ◽

In Vitro System ◽

Scanning Electron

SummaryA technique for harvesting mesothelial cells is described. This entails collagenase digestion of omentum after which the cells can be cultured. The technique has been developed using the rat, but has also been successfully applied to human tissue. Cultured rat mesothelial cells obtained in this way have been examined by scanning electron microscopy. Rat mesothelial cells grown on plastic film have been exposed to blood in an in vitro system using a Baumgartner chamber and have been demonstrated to support blood flow. No adhering platelets were observed on the mesothelial cell surface. Fibroblasts similarily exposed to blood as a control were washed off the plastic.

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